Human antibiotic proteins

ABSTRACT

The invention relates to proteins, notably SAP-2 and SAP-3, having an antibiotic action. The invention also relates to a method for purifying certain antimicrobial proteins, as well as to a use of said antimicrobial proteins for antibiotic therapy or to a use of cells which were transfected with a DNA which codes for the proteins provided for in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of, and claims priority from, U.S. Pat.No. 7,419,781, filed Jan. 22, 2002, which is the U.S. National Stage ofInternational Application No. PCT/EP2000/00776, filed Feb. 1, 2000,which claims benefit of German patent applications, 19949436.3, filedOct. 8, 1999 and 19905128.3, filed Feb. 1, 1999.

BACKGROUND

The invention relates to proteins/peptides (proteins), which have anantibiotic action. In addition, the invention comprises a process forthe purification of certain antibiotic proteins. The invention alsorelates to a use of the proteins for antibiotic treatment or to a use ofcells that were transfixed with a DNA that codes for the antibioticproteins.

PRIOR ART

Pathogenic microorganisms are usually found on the surfaces ofepithelial cells. The microorganisms adhere to the cells and arereproduced. They also sometimes penetrate the deeper tissue layers.Since the immunological response to these pathogenic microorganisms setsin slowly, it is not surprising that the epithelial cells have a defenseto take action against the microorganisms with the aid of secretedantimicrobial substances. Some of these substances lead to malnutritionin the microorganisms; others kill the microorganisms by the structuresof the microorganisms being destroyed.

The epithelial cells of mammals are not infected in the normal way.Nevertheless, the skin surface is densely populated by bacteria andfungi. In this case, these are skin-specific microorganisms that, ifthey are under control, are not pathogenic.

The first known epithelial β-defensin, which protects the trachea ofbovines, is TAP, which has 64 amino acids. (D. G. ZASLOFF et al. (1991)Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 3952-3956). This bovine TAP hasan antibacterial action against E. coli, Klebsiella pneumoniae,Staphylococcus aureus and Pseudomonas aeruginosa at a minimum inhibitionconcentration of 12-50 μg/ml. Candida albicans is also destroyed. Inthis case, this is a protein that is expressed in a tissue-specificmanner.

Another β-defensin protects the tongues of bovine animals, namely LAP,which is highly homologous to TAP. (B. S. SCHONWETTER et al. (1995)Science Vol. 267, pp. 1645-1648).

Not until 1995 was the first human β-defensin found, which is namedhBD-1. (K. W. BENSCH et al. (1995) FEBS Lett., Vol. 368, pp. 331-335).hBD-1 thus has an antibacterial action again gram-negative bacteria at aconcentration of 60 to 500 μg/ml. (M. GOLDMAN et al. (1997) Cell. Vol.88, pp. 553-560). hBD-1 is expressed in a dominant manner in thekidneys, but other epithelial tissues also secrete hBD-1.

Regardless of these studies, there was interest in what normally keepsthe skin healthy and why the skin is seldom infected. The secondβ-defensin that was isolated in humans was the hBD-2, which consists of41 amino acids. (J. HARDER et al. (1997) Nature, Vol. 387, p. 861).hBD-2 acted very effectively against gram-negative bacteria with an LD₉₀of 10 μg/ml; conversely, in the case of gram-positive bacteria, thevalue exceeds 100 μg/ml. The hBD-2 is an inducible peptide, which canitself be induced by some heat-inactivated bacteria.

Another human antibiotic protein is the ALP, a protease inhibitor (J. A.KRAMPS et al. (1988) Biol. Chem. Hoppe Seyler, Vol. 369, pp. 83-87),which is produced by keratinocytes and directed against several bacteriaand fungi.

The attacks of antibiotic proteins are very different. Interaction withthe membranes of the microorganisms are common. Lipophilic structures ofmany antibiotic proteins and the defensins speak for intercalation inthe membranes or penetration of the membranes. The antimicrobialproteins and peptides have a toxic action only in the microorganismsthemselves.

OBJECTS AND SOLUTION

The object of the invention is to offer additional human, antibioticproteins and their derivatives, which can be used effectively againstmicroorganisms, especially against gram-negative and gram-positivebacteria, against fungi and against viruses.

Sequences of Mature Proteins

The object is achieved by at least one protein,

-   -   a) which has one of the following sequences as an active, mature        protein/peptide (protein):        -   (i) SEQ ID NO: 1 (Sequence Protocol No. 1) (SAP-2); or        -   (ii) SEQ ID NO: 2 (Sequence Protocol No. 2) (SAP-3);    -   or    -   b) which has allelic modifications of one of the amino acid        sequences that are mentioned above under a) as an active, mature        protein, whereby at least one amino acid of the amino acid        sequence is substituted, deleted, or inserted, without in this        case significantly affecting the activity of the active protein,    -   or    -   c) which has post-translational modifications of one of the        sequences under a) and b) as an active, mature protein, and        these modifications do not significantly affect the activity of        the active protein.

A protein according to the invention that has an antimicrobial and/orantibiotic action is advantageous.

A protein according to the invention that has an antimicrobial orantibiotic action and that has a mobility of 6 kDa in the SDS-gelelectrophoresis is advantageous.

More preferred is a protein according to the invention that has anantibiotic activity against Escherichia coli or Staph. aureus at aconcentration of less than 100 μg/ml.

In the literature, the designation SAP-2 is to be replaced by RNase 7 inthe future, and the term SAP-3 is to be substituted by hBD-3 in thefuture.

Very preferred is a protein according to the invention that is a proteinthat is provided with the human amino acid sequence (cf. SEQ ID NO: 1 to2).

Regarding SEQ ID NO: 1

All allelic modifications that comprise the substitutions, the deletionsand/or the insertions of up to 30 amino acids belong to the group ofproteins of SEQ ID NO: 1 according to the invention. Preferred aredeletions, substitutions and/or insertions of up to 20 amino acids, andmore preferred are those of up to 10 amino acids; most preferred are thedeletions, substitutions and/or insertions of one, two, three, four,five, six, seven, eight or nine amino acids. The allelic modificationsare not limited to the naturally occurring alleles, rather changes ofthe amino acid sequence that are produced in the laboratory (notoccurring in nature itself) are also possible.

Regarding SEQ ID NO: 2

All allelic modifications that comprise the substitutions, the deletionsand/or the insertions of up to 10 amino acids belong to the group ofproteins of SEQ ID NO: 2 according to the invention. Preferred aredeletions, substitutions and/or insertions of up to 6 amino acids, andmore preferred are those of up to 4 amino acids; most preferred aredeletions, substitutions and/or insertions of one, two or three aminoacids. The above-mentioned broadening to changes that comprisesynthetically producible changes in addition to naturally occurringchanges also applies here.

Sequences of Mature Proteins with Signal Sequence

The object is also achieved by at least one protein, which comprises asignal sequence and a mature protein according to the invention,

-   -   d) whereby the protein has one of the following sequences:        -   (i) SEQ ID NO: 3 (PreSAP-2); or        -   (ii) SEQ ID NO: 4 (PreSAP-3);    -   or    -   e) whereby the protein has allelic modifications of one of the        amino acid sequences that are mentioned above under d), whereby        at least one amino acid of the amino acid sequence is        substituted, deleted or inserted, without in this case        significantly affecting the activity of the mature active        protein,    -   or    -   f) whereby the protein has post-translational modifications of        one of the sequences under d) and e), which do not significantly        affect the activity of the active mature protein.        Regarding SEQ ID NO: 3

All allelic modifications that comprise the substitutions, the deletionsand/or the insertions of up to 35 amino acids belong to the group ofproteins of SEQ ID NO: 3 according to the invention. Preferred aredeletions, substitutions and/or insertions of up to 23 amino acids, andmore preferred are those of up to 12 amino acids; most preferred aredeletions, substitutions and/or insertions of one, two, three, four,five, six, seven, eight or nine amino acids. The allelic modificationsare not limited to the naturally occurring alleles, but rather changesof the amino acid sequence that are produced in the laboratory (notoccurring in nature itself) are also possible.

Regarding SEQ ID NO: 4

All allelic modifications that comprise the substitutions, the deletionsand/or the insertions of up to 13 amino acids belong to the group ofproteins of SEQ ID NO: 4 according to the invention. Preferred aredeletions, substitutions and/or insertions of up to 8 amino acids, andmore preferred are those of up to 6 amino acids; most preferred aredeletions, substitutions and/or insertions of one, two, three, four orfive amino acids. The above-mentioned broadening to changes thatcomprise synthetically producible changes in addition to naturallyoccurring changes also applies here.

Most preferred is a protein according to the invention that is arecombinant protein. In this case, the proteins can be glycosylated ifthis is a SAP-2 or a variant thereof.

The proteins according to the invention comprise the mature proteins andthe corresponding precursor proteins, which consist of a signal sequenceand the sequence of the mature protein. In this case, the signalsequence presupposes the sequence of the mature protein. The matureprotein begins with the above-mentioned N-terminal sequence under itema). The signal sequence is necessary for the penetration of theendoplasmatic reticulum.

It is also possible to synthesize protective groups, which are knownfrom peptide chemistry, at the N-terminus and/or C-terminus.

The protective group of the N-terminus can consist of:

Alkyl, aryl, alkylaryl, aralkyl, alkylcarbonyl or arylcarbonyl groupswith 1 to 10 carbon atoms; preferred are naphthoyl, naphthylacetyl,naphthylpropionyl, benzoyl groups or an acyl group with 1 to 7 carbonatoms.

The protective group of the C-terminus can consist of:

A substituted or unsubstituted alkoxy or aryloxy group with 1 to 10carbon atoms or an amino group.

Other protective groups—both for the N-terminus and for theC-terminus—are described in Houben-Weyl (1974) Georg Thieme Verlag, 4thEdition. The description of the protective groups in the citedbibliography is part of the disclosure.

The sequence of the protein according to the invention can be connectedwith other framework-amino acid sequences (analogously to the definitionof “framework” in antibodies) at the N-terminal and/or C-terminal endinstead of a protective group. These other framework-amino acidsequences are not essential for the bonding of the protein according tothe invention, but they can be vehicles of other functions, and thusinclude, for example, chelates or else cytostatic or cytotoxicsequences. Such framework-amino acid sequences occur in nature. Thesemay be, for example, the sequences of the variable area of an antibodythat are arranged between the hypervariable areas. These sequences arereferred to as “framework” (framework sequences). As framework-aminoacid sequences, non-cleaved partial signal sequences of a secretedeukaryotic protein are also known, whereby the protein is expressed in abacterium. At times, such signal sequences have no effect on thefunction of the subsequent protein. It is also possible to coupleproteins according to the invention behind one another, wherebyframework-amino acid sequences are arranged between the individualsequences.

To decide in individual cases whether a certain protein according to theinvention with at least one framework-amino acid sequence and/or atleast one protective group is to be included in the subject of theinvention, a comparison can be made between

-   -   (i) this protein with a framework-amino acid sequence and/or        with a protective group, and    -   (ii) the same protein without a framework-amino acid sequence        and without a protective group.        In this case, the two molecules that are compared should have        essentially the same functions of inhibition or binding.        cDNA or DNA that Code for the Proteins According to the        Invention

The invention also comprises a cDNA or DNA,

-   -   aa) whereby the cDNA or DNA codes one of the following amino        acid sequences:        -   (i) SEQ ID NO: 1 (SAP-2);        -   (ii) SEQ ID NO: 2 (SAP-3);        -   (iii) SEQ ID NO: 3 (PreSAP-2); or        -   (iv) SEQ ID NO: 4 (PreSAP-3)    -   or    -   bb) whereby the cDNA or DNA codes allelic modifications of one        of the amino acid sequences under aa),        -   in which at least one amino acid of the amino acid sequence            is substituted, deleted or inserted, without in this case            significantly affecting the activity of the active protein.

cDNA and DNA, which code a mature protein according to the invention,are preferred.

The allelic modifications have been defined above under the item“Sequences of the Mature Proteins.”

Regarding SEQ ID NO: 1 and 3

All allelic modifications that comprise the substitutions, the deletionsand/or the insertions of up to 90 nucleotides belong to the group ofDNAs of SEQ ID NO: 1 and 3 according to the invention. Preferred aredeletions, substitutions and/or insertions of up to 60 nucleotides, andmore preferred are those of up to 30 nucleotides; most preferred aredeletions, substitutions and/or insertions of one, two, three, four,five, six, seven, eight or nine or 10 to 29 nucleotides. The allelicmodifications are not limited to the naturally occurring alleles, butrather changes of the amino acid sequence that are produced in thelaboratory (not occurring in nature itself) are also possible.

Regarding SEQ ID NO: 2 and 4

All allelic modifications that comprise the substitutions, the deletionsand/or the insertions of up to 30 nucleotides belong to the group ofDNAs of SEQ ID NO: 2 and 4 according to the invention. Preferred aredeletions, substitutions and/or insertions of up to 18 nucleotides, andmore preferred are those of up to 12 nucleotides; most preferred aredeletions, substitutions and/or insertions of one, two, or three or 4 to11 nucleotides. The above-mentioned broadening to changes that comprisesynthetically producible changes in addition to naturally occurringchanges also applies here.

In addition, the invention comprises a cDNA or DNA,

-   -   cc) whereby the cDNA or DNA has one of the following nucleotide        sequences:        -   (i) SEQ ID NO: 5 (cDNA-SAP-2)        -   (ii) SEQ ID NO: 6; (cDNA-SAP-3);    -   or    -   dd) whereby the cDNA or DNA has an allelic modification of one        of the nucleotide sequences under cc), whereby at least one        nucleotide is substituted, deleted or inserted, without in this        case significantly affecting the activity of the protein, which        is coded by the allelic modification of the nucleotide sequence        under cc).        Regarding SEQ ID NO: 5

All allelic modifications that comprise the substitutions, the deletionsand/or the insertions of up to 90 nucleotides belong to the group ofDNAs of SEQ ID NO: 5 according to the invention. Preferred aredeletions, substitutions and/or insertions of up to 60 nucleotides, andmore preferred are those of up to 30 nucleotides; most preferred aredeletions, substitutions and/or insertions of one, two, three, four,five, six, seven, eight or nine or 10 to 29 nucleotides. The allelicmodifications are not limited to the naturally occurring alleles.

Regarding SEQ ID NO: 6

All allelic modifications that comprise the substitutions, the deletionsand/or the insertions of up to 30 nucleotides belong to the group ofDNAs of SEQ ID NO: 6 according to the invention. Preferred aredeletions, substitutions and/or insertions of up to 18 nucleotides, andmore preferred are those of up to 12 nucleotides; most preferred aredeletions, substitutions and/or insertions of one, two, or three or 4 to11 nucleotides. The above-mentioned broadening to changes that comprisesynthetically producible changes in addition to naturally occurringchanges also applies here.

Preferred are cDNA and DNA, which code a protein according to theinvention.

Another embodiment of the invention comprises a cDNA or DNA,

-   -   ee) whereby the cDNA or DNA has one of the following nucleotide        sequences:        -   (i) SEQ ID NO: 7 (cDNA-PreSAP-2) or        -   (ii) SEQ ID NO: 8 (cDNA-PreSAP-3),    -   or    -   ff) whereby the cDNA or DNA has an allelic modification of one        of the nucleotide sequences under ee), whereby at least one        nucleotide is substituted, deleted or inserted, without in this        case significantly affecting the activity of the protein, which        is coded by the allelic modification of the nucleotide sequence        under ee).

cDNA and DNA, which code a preprotein according to the invention, arepreferred.

Regarding SEQ ID NO: 7

All allelic modifications that comprise the substitutions, the deletionsand/or the insertions of up to 90 nucleotides belong to the group ofDNAs of SEQ ID NO: 5 according to the invention. Preferred aredeletions, substitutions and/or insertions of up to 60 nucleotides, andmore preferred are those of up to 30 nucleotides; most preferred aredeletions, substitutions and/or insertions of one, two, three, four,five, six, seven, eight or nine or 10 to 29 nucleotides. The allelicmodifications are not limited to the naturally occurring alleles.

Regarding SEQ ID NO: 8

All allelic modifications that comprise the substitutions, the deletionsand/or the insertions of up to 30 nucleotides belong to the group ofDNAs of SEQ ID NO: 6 according to the invention. Preferred aredeletions, substitutions and/or insertions of up to 18 nucleotides, andmore preferred are those of up to 12 nucleotides; most preferred aredeletions, substitutions and/or insertions of one, two, or three or 4 to11 nucleotides. The above-mentioned broadening to changes that comprisesynthetically producible changes in addition to naturally occurringchanges also applies here.

All DNA constructs also then include the listed sequences according tothe invention, if such nucleotides are exchanged, which code the sameamino acid based on the degenerated code. The exchange of suchnucleotides is obvious, and the corresponding amino acids are disclosedin any biochemistry textbook. (R. KNIPPERS, 1982, 3rd Edition,Molekulare Genetik [Molecular Genetics], Georg Thieme Verlag)

The allelic modifications have been defined above.

If the activity of the protein is indicated to determine whether theallelic modification is included under the group of the proteinsaccording to the invention, the mature protein is thus always to bemeasured, even if the signal sequence is also indicated. If the signalsequence should be indicated, the function is always to be measured atthe protein, which is obtained after the signal sequence is removed.

The activity of the proteins according to the invention is measured interms of its function, which can be an antibiotic action, anantimicrobial action and/or the binding to antibodies or bindingmolecules that are directed against the mature human protein.

The invention also comprises binding molecules (for example peptides orderivatives thereof), single-chain proteins, antibodies or fragments ofantibodies, which specifically detect domains on the mature proteinaccording to the invention. If the purified protein according to theinvention is present, it is easily possible for one skilled in the artto produce monoclonal antibodies. In this case, the known method ofKöhler and Milstein and its extensions are used. In particular, inconventional methods, a mouse is immunized several times with thepurified protein, the splenocytes are removed and fused with suitabletumor cells. The hybrids are then selected. The binding molecules can beused as a diagnostic agent to determine, for example, whether therespective patient suffers from a deficiency or a variant of theproteins according to the invention.

The proteins of the invention can be isolated from, for example, hornyscales of psoriasis patients. Purification is done according to theexamples. The proteins have the above-described amino acid sequences.They have a molecular weight of about 20,000±2,000 with SAP-2 and6,000±2,000 with SAP-3 (see examples). The isoelectric point lies in arange of pH 8.5 to 10.5, if the method that is described in the exampleis used.

The proteins according to the invention can have a natural origin. Theproteins are obtained by being harvested and worked up according to theexamples. The horny scale supernatant is purified, and the proteinsaccording to the invention are isolated and concentrated. Allconcentration stages of the isolation and the purification are part ofthe invention. Preferred are the concentration stages of isolation andpurification, in which the proteins according to the invention can beused for pharmaceutical purposes. Purifications of 50% of the proteinsrelative to the total protein thus are achieved; preferred are 85%, morepreferred 95% and most preferred 99% of the proteins relative to thetotal protein.

It is also possible to produce the proteins according to the inventionsynthetically. This includes protein synthesis according to J. M. SEWARTand J. D. YOUNG, San Francisco, 1969 and J. MEIENHOFER, HormonalProteins and Peptides, Vol. 2, p. 46, Academic Press (New York), 1973and E. SCHODER and K. LUBKE, The Peptides, Vol. 1, Academic Press (NewYork) 1965. The citations are part of the disclosure.

The synthetically produced proteins also include the recombinantproteins, which are produced according to known processes. Depending onthe host organism, the proteins according to the invention (with SAP-2)can be glycosylated or, if they are synthesized in prokaryotes,unglycosylated.

The function of toxicity against microorganisms can be determined invarious testing systems. In the examples, standard testing proceduresare described. (Cf. SELSTED et al. (1993) J. Biol. Chem., Vol. 268, pp.6641-6648 and GANZ et al. (1985) J. Clin. Invest. Vol. 76, pp.1427-1435)

The proteins of the invention have an antibiotic action againstmicroorganisms, especially against the gram-negative and gram-positivefamilies, in this case preferably against the E. coli and Staphylococcusaureus types.

The testing systems are described in detail in Example 3.

Vectors with the DNA According to the Invention

Another part of the invention is a vector that contains a cDNA or DNAaccording to the invention, also a suitable promoter and optionally asuitable enhancer. A signal sequence can also be comprised. Vectors aredescribed in more detail in European publications EP 0 480 651, EP 0 462632 and EP 0 173 177.

Another embodiment of the invention consists in a eukaryotic orprokaryotic host cell, which is transformed with a vector according tothe invention.

The invention also comprises a process for the production of a proteinaccording to the invention with use of a host cell according to theinvention, with the steps:

Cultivation of the host cell,

accumulation of the protein, and

purification of the protein.

The invention also comprises a process for synthesizing one of theproteins according to the invention, whereby the proteins aresynthesized according to the solid-phase method or according to theliquid-phase method.

The process in which the proteins according to the invention areproduced has the following stages:

The carboxyl end of an amino acid that is to be coupled, its aminogroups and optionally functional groups of the side chain carry aprotective group, reacts with the free amino end of the amino acid thatis to be coupled or the protein fragment that is to be coupled in thepresence of a condensation reagent, and

-   -   in the case of a non-terminal amino acid, the α-amino protective        group is then cleaved off from the coupled amino acid, and other        amino acids are coupled to the protein chain that is to be        synthesized according to the two steps that are described above,

or

-   -   in the case of a terminal amino acid, the α-amino protective        group is optionally then cleaved off from the coupled amino acid        and

after the last amino acid is coupled in the case of the solid-phasemethod, the protein is cleaved off from the solid phase.

Allelic Modifications

Most deletions, insertions and substitutions do not appear to result inany drastic change in the characteristics of the protein of theinvention. Since it is difficult to indicate the exact effects of asubstitution, a deletion or an insertion beforehand, the function of thealtered protein must be compared with the function of the proteinaccording to the invention. The methods that are to be used for thispurpose are indicated in the examples. As a standard, the proteinaccording to SEQ ID NO: 1 to 2 is used, and the protein that is purifiedaccording to Example 1 or 2 and also the purification methods of Example1 or 2 are used for the comparison protein.

The genetic code is degenerated, i.e., most amino acids are coded bymore than one codon that consists of three nucleotides. Several allelicmodifications on the plane of the nucleotides therefore do not lead toan alteration of the amino acid sequence. Allelic modificationstherefore take place in particular on the plane of DNA and can have asecondary effect on the amino acid sequence.

The cDNA or DNA sequences, which code the proteins according to theinvention, can be modified according to conventional techniques toproduce variants of the proteins according to the invention, whichessentially have the same activity as the proteins of the invention thatare described and characterized. In this case, the activity is measuredas it is described in the examples. Such a complete homology testing isdescribed in CUNNINGHAM et al. (1989) Science, Vol. 243, p. 1330, andO'DOWD et al. (1988), J. Biol. Chem., Vol. 263, p. 15985.

Amino acids can be substituted, whereby the amino acids can besubstituted in their positions with a protein or peptide mapping,whereby then the activity of the modification is measured. In this case,substitutions that are determined by experiment are possible, which isnot easily predictable owing to the chemical structure of the sidechains.

The mutations are defined by the homology (similarity) of two proteinspending comparison. The term homology comprises similar amino acids andgaps in the sequences of the amino acids (homology=similarity). Theproteins according to the invention have amino acid sequences, whichhave a homology of at least 80%, preferably 90%, more preferably 95% andmost preferably 98% of the structures according to the invention, asthey are defined by the sequences under SEQ ID NO: 1 to 2 or SEQ ID NO:3 and 4 and as they are also obtained after purification according tothe examples.

Sequences of proteins can be simply changed. In this case, the aminoacids are exchanged in their respective positions. At the same time, itis necessary to study the function of the thus obtained sequences. Theamino-acid exchange can be carried out according to two differentmethods.

Each position of a protein is replaced in succession by alanine. Then,the function of the molecule that is modified in each case by alanine ismeasured. If the measured value deviates from that of the standardprotein, this amino acid at this position of the protein, on which analanine is now arranged, is essential for the function. To this end, amap of the protein, from which the conservative positions and thepositions that are accessible to a variation are indicated, is produced.

Another method consists in exchanging each position or essentialpositions of a protein for all 20 natural amino acids. Then, thefunction is tested by all of these modifications. The method isdescribed in Ronald FRANK (1992) Spot Synthesis: Easy Technique for thePositionally Addressable, Parallel Chemical Synthesis on a MembraneSupport. Tetrahedron Vol. 48, No. 42, pp. 9217-9232.

Both methods are especially suitable for peptides, since the latter areproduced with the solid-phase synthesis. Nevertheless, these methods caneasily also be transferred to proteins for one skilled in the art,whereby the proteins are synthesized in cells. To this end, analteration of the DNA is essential, which can take place specifically,however, with the present techniques.

The process for modifying the amino-acid sequence can be explained asfollows:

-   -   (a) At least one amino acid in the sequence of the protein is        replaced by a natural amino acid or else optionally by an amino        acid that is not natural.    -   (b) The modified protein is tested after each substitution on        the antibiotic function relative to microorganisms, and the most        antibiotic proteins are selected.    -   (c) In another step, the most antibiotic proteins are run at        least in another cycle according to items (a) and (b).

The result of such a modification can be a protein that only has someparts in common with the original sequence.

As mentioned above, the invention also comprises modifications of theDNA or cDNA. These modified sequences hybridize under stringentconditions with the DNA sequences that code the proteins according tothe invention (see sequences under aa); cc) and ee)). The cDNA- or DNAsequences have nucleotide sequences, which have an identity includingshorter (up to 15 nucleotides) deletions and insertions of at least 70%,preferably 82%, more preferably 90% and most preferably 95% with thecDNA or DNA sequences according to the invention (see aa), cc) and ee)).The identity including the short (up to 15 nucleotides) deletions andinsertions can be measured by hybridization, as it is described in R.KNIPPERS, Molekulare Genetik, 1982, Third Edition, Georg Thieme VerlagStuttgart, New York. In addition, standard computer programs are knownto one skilled in the art, with whose help homology can be calculated.

The invention also comprises a cDNA or DNA with at least one of thesequences of SEQ ID NO: 5 to 8, or nucleotide sequences, which hybridizewith one of SEQ ID NO: 5 to 8 under selective, stringent conditions.

Stringent conditions are then present if the salts, theirconcentrations, the temperature of the inorganic and organic solventsare controlled in typical form, as is practiced in the establishedhybridization technique. Stringent temperature conditions includetemperatures of at least 30° C., preferably at least 37° C., morepreferably at least 45° C., still more preferably at least 55° C., stillmore advantageously at least 65° C. and most preferably at least 70° C.Stringent salt concentrations comprise less than 1000 mmol, preferablyless than 700 mmol, more preferably less than 400 mmol, still morepreferably less than 300 mmol, advantageously less than 200 mmol andmost preferably 150 mmol. The combination of the parameters is moreimportant than the reference to an individual parameter. (WETMUR et al.(1968) J. Mol. Biol., Vol. 31, p. 349)

Post-Translational Modifications

The above-mentioned post-translational modifications are defined aschanges that occur during or after translation. These include theglycosylation, the formation of disulfide bridges, the chemicalmodifications of amino acids, thus, for example, the sulfation, which isdescribed in connection with hirudin. (J. W. FENTON (1989) “ThrombinInteractions with Hirudin,” Seminars in Thrombosis and Hemostasis, Vol.15, pp. 265-268)

The glycosylation is a basic function of the endoplasmatic reticulumand/or the Golgi apparatus. The sequence and the branching of theoligosaccharides is formed in the endoplasmatic reticulum and altered inthe Golgi apparatus. The oligosaccharides can be N-linkedoligosaccharides (asparagine-linked) or O-linked oligosaccharides(serine-, threonine- or hydroxylysine-linked). The form of glycosylationdepends on the producing cell type and on the type from which thecorresponding cell type is derived. The extent and the type ofglycosylation can be affected by substances, as it is described inEuropean Publication EP 0 222 313. The variation of the glycosylationcan alter the function of the protein.

Proteins form frequently covalent bonds within the chains. Thesedisulfide bridges are produced between two cysteines. In this case, theprotein is specifically precipitated. The disulfide bridges stabilizethe three-dimensional structure of the proteins.

Isolation and Production of the Proteins According to the Invention

The invention also comprises a process for the purification of proteinsaccording to the invention, whereby the process consists of thefollowing steps:

-   -   (i) Extraction of proteins from natural human epithelial cells,        transfixed cells or skin scales or cell cultures, which were        optionally exposed to microorganisms,    -   (ii) Application of the extract on an affinity column with        subsequent Reversed Phase HPLC and elution via a salt gradient,        with acids or organic eluents,    -   or    -   (iii) Application of the extract on an HPLC column and elution        with salts.    -   The purification is described in detail in the examples.

Preferred is a micro-mono S-HPLC column.

The proteins are preferably purified according to Examples 1 and 2.Other isolation and purification methods are also possible, however:

-   Methods of Enzymology, Volume 182: Guide to Protein Purification,    ed. Murray P. DEUTSCHER, Academic Press, 1990;-   Protein Purification Application—A Practical Approach, ed. E. L. V.    HARRIS and S. ANGEL, IRL Press, 1990;-   Protein Purification, Principles and Practice, Ropert SCOPES,    Springer-Verlag 1982; and-   Protein Purification, Principles, High Resolution Methods and    Applications, ed. H.-C. JANSON and L. RYDEN, VCH Publishers 1989.

Use as Pharmaceutical Agents

The proteins according to the invention have pharmacological effects andcan therefore be used as pharmaceutical active ingredients. Theinvention also comprises a pharmaceutical agent that contains one of theproteins according to the invention or a mixture thereof. Apharmaceutical composition, which contains one of the proteins accordingto the invention or a mixture of proteins according to the invention, inthe presence of pharmaceutically compatible and acceptable compounds andvehicles, is also a part of the invention. The invention also comprisesa pharmaceutical composition that contains one of the pharmaceuticallyactive proteins according to the invention or mixture thereof and apharmaceutically compatible salt or a pharmaceutically compatiblevehicle.

The proteins according to the invention according to SEQ ID NO: 1 to 2in particular show a toxic or antibiotic action with regard tomicroorganisms, especially with regard to the groups of gram-negativeand gram-positive bacteria, preferably in the case of the E. coli andSt. aureus types. Testing procedures are described in Example 3.

The test results of this in vitro test show that the proteins accordingto the invention can be used as pharmaceutical agents or for medicaltreatment. These test results can be transferred from the in vitro testsystem to an in vivo system, since these are established testingarrangements in the tests. The proteins of the invention can thereforebe used for treatment and prevention of infections by microorganisms.The proteins of the invention can be used as an antibiotic medication inmammals, especially humans, for treating infections and/or for infectionprophylaxis.

The invention additionally provides

-   -   (i) the use of one of the proteins according to the invention or        mixture thereof for the production of a medication for treatment        of infections that were caused by microorganisms or for        prevention of such infections;    -   (ii) a process for treating infections that were caused by        microorganisms or for prevention of such infections, said        process comprises an administration of an amount of protein        according to the invention, whereby the amount suppresses the        disease, and whereby the amount of protein is given to a patient        who requires such a medication;    -   (iii) a pharmaceutical composition for treating infections that        were caused by microorganisms or for prevention of such        infections, said treatment comprises one of the proteins        according to the invention or mixture thereof and at least one        pharmaceutically compatible vehicle and additive.

For this therapeutic action, different doses are suitable. They dependon, for example, the protein that is used, the host, the type ofadministration and the type and difficulty of the conditions that are tobe treated. In general, however, satisfactory results are to be expectedin animals if the daily doses comprise a range of 2 μg to 2000 μg per kgof body weight. In the case of larger mammals, for example humans, arecommended daily dose lies in the range of 2 to 2000 μg per kg of bodyweight, if the protein that is purified according to Example 1 or 2 isused. For example, this dose is suitably administered in partial dosesup to four times daily. The daily dose in the case of prevention is atenth of the amount that is used in the case of an infection.

The protein according to the invention is preferably locally orepithelially administered, thus also in the upper and lower airpassages.

The proteins according to the invention can be administered in anycommonly used method, also in the form of creams, gels, semisolid dosageforms, suspensions or inhalational solutions or inhalational powders.

This invention makes available pharmaceutical compositions that compriseone of the proteins according to the invention or mixture thereof and atleast one pharmaceutically compatible vehicle or additive. Suchcompositions can be produced according to known processes. In this case,reference is to be made to Remington's Pharmaceutical Science, 15th Ed.Mack Publishing Company, East Pennsylvania (1980).

In addition, syngeneic or allogeneic human cells that are transfixedwith DNA or cDNA according to the invention can be used as medication,by these cells being applied to the epithelial tissue or being locatedin the matrix of a bandage.

DEFINITIONS

-   -   “Antimicrobial” means that the proteins according to the        invention        -   (i) inhibit and/or prevent the growth and/or the            proliferation of microorganisms and/or        -   (ii) destroy the microorganisms or structures thereof.    -   “Antibiotic” means that the proteins according to the invention        have an adverse effect on the normal biological function of the        microorganisms, whereby this means death or destruction, as well        as inhibition of growth or proliferation of the microorganisms,        along with impairment of metabolic functions. Antibiotic also        comprises the term antimicrobial. An antibiotic action can also        be present in viruses. Antibiotic therefore also comprises        antiviral.    -   “Antiviral” means that DNA and RNA viruses can be controlled        with the aid of the proteins of the invention. In this case,        various possible interventions are useful. The viruses can be        influenced in their dormant forms. The adhesion phase to the        host or the penetration in the host can be destroyed, and the        retention or the reproduction (temperent or virulent phase) can        be impaired in the host.    -   The proteins according to the invention can also be used for        healing wounds.    -   “Healing wounds” means that, for example, the contraction of        wounds is accelerated, that connective tissue has begun to form        in the wound area, that collagen is stored. Burns can also be        treated well with the proteins of the invention. In this case, a        bandage can be enriched with proteins or proteins of special,        especially transfixed cells can be expressed in the bandage.    -   “Microorganisms” comprise the prokaryotes with eubacteria and        archaebacteria, fungi (mycota with myxomycetes, phycomycetes and        eumycetes), plant and animal protozoan organisms and viruses.    -   The term “proteinT” comprises all lengths of amino acid        sequences, thus also peptides. In this case, proteins can also        consist of various chains, which are connected by covalent bonds        or van der Waal's forces.

Combination with Antibiotics

The proteins according to the invention can be administered togetherwith antibiotics, for example, from the following group: bacitracin,gramicidin, polymyxin, vancomycin, teichoplanin, aminoglycosides,penicillin, and monobactam.

Diagnostic Agent

The invention also comprises the use of at least one protein accordingto the invention for the production of antibodies or fragments thereof.

The invention also comprises the use of an antibody according to theinvention or fragments thereof as a diagnostic agent.

In this case, the proteins according to the invention are to be detectedin body tissues and bodily fluids. The detection processes can thus alsobe produced by coupling ligands to the proteins according to theinvention.

EXAMPLES Example 1 Extraction of SAP-2 1.1 Isolation

50 g of lesional psoriasis scales was extracted under acid conditions inthe presence of ethanol and concentrated by evaporation. In this case,the process was followed that is described in J. M. SCHRÖDER, (1997)Methods in Enzymology, Vol. 288, pp. 266-296.

After diafiltration on 0.02 mol/l of sodium phosphate buffer, pH 8 andcentrifuging, the supernatant was chromatographed on a bacteria-affinitycolumn (E. coli or Staph. aureus), which had been produced by couplingheat-inactivated (70° C. over one hour) E. coli or Staphylococcus aureusbacteria to an N-hydroxy-succinimide-activated sepharose column(Pharmacia) (10×5 mm).

The column was washed first with the equilibration buffer, and thenbonded proteins were eluted with an acid buffer (0.1 mol/l of glycinebuffer, pH 3 with 1 mol/l of NaCl).

The eluate that contains bonded protein was diafiltered from 0.1% ofaqueous trifluoroacetic acid solution and first subjected to apreparative Reversed Phase HPLC separation analogously to isolation fromchemotactic peptides (cf. J. M. SCHRÖDER, (1997) Methods in Enzymology,Vol. 288 pp. 266-296). 20 μl of the respective fractions wasfreeze-dried, taken up in 5 μl of a 0.01 percent aqueous acetic acidsolution and analyzed with the aid of a plate diffusion test system (seeExample 3) (for the identification of antimicrobial or antibioticproteins) analogously to M. E. SELSTED et al. (1993) J. Biol. Chem.,Vol. 268, pp. 6641-6648 with respect to the presence of antimicrobialpeptides (with Staph. aureus and E. coli as test bacteria).

Antimicrobially active proteins that were eluted with 40% acetonitrilewere then subjected—analogously to the isolation of chemotactic peptides(J. M. SCHRÖDER, (1997) Methods in Enzymology, Vol. 288, pp. 266-296)—toa micro-mono S-HPLC separation with the aid of the Smart-HPLC system.

SAP-2 was eluted with 0.8 mol/l of NaCl.

A subsequent micro-reversed phase-HPLC analysis with the aid of a C-18RP column yielded a protein peak that is eluted with 52% acetonitrile,which after SDS-gel electrophorese (performed according to the method ofJ. M. SCHRÖDER, (1997) Methods in Enzymology, Vol. 288, pp. 266-296),yielded an individual protein band or two bands corresponding to themobility of about 20 kDa.

1.2. Sequencing of Fragments

Sequencing tests yielded the amino-terminal sequence with the numberingfrom the complete sequence

Pro Lys Gly Met Thr Ser Ser Gln Trp Phe Lys Ile             5                   10Gln His Met Gln Pro Ser Pro Gln Ala Cys Asn Ser     15                  20                  25Ala Met Lys Asn Ile Asn Lys His Thr Lys Arg Cys                 30                  35 Lys Asp(amino acid residues 2-39 of SEQ ID NO: 1)

The Edman degradation of the peptide fragment yielded a sequence thatcorresponds to the C-terminus:

Asp Ser Gln Gln Phe His Leu Val Pro Val His Leu        115                 120 Asp Arg Val Leu 125(amino acid residues 113-128 of SEQ ID NO: 1)

1.3. Biological Activity of SAP-2 MIC

Antimicrobial activity against Staph aureus: <100 μg/ml

-   -   against E. coli <50 μg/ml

RNase activity: 1.2 μg of SAP-2 digested about 5 μg of human RNA in onehour at 37° C.

Example 2 Extraction of SAP-3 2.1: Isolation

50 g of lesional psoriasis scales was extracted under acid conditions inthe presence of ethanol and concentrated by evaporation. In this case,the process was followed that is described in J. M. SCHRÖDER, (1997)Methods in Enzymology, Vol. 288, pp. 266-296.

After diafiltration on 0.02 mol/l of sodium phosphate buffer, pH 8 andcentrifuging, the supernatant was chromatographed on an anti-IL-8affinity column, which had been produced by coupling monoclonalanti-IL-8 antibody 52E4 to an N-hydroxy-succinimide-activated sepharosecolumn (Pharmacia) (analogously to J. M. SCHRÖDER, (1997) Methods inEnzymology, Vol. 287, pp. 216-230).

The column was washed first with the equilibration buffer, and thenbonded protein was eluted with an acid buffer (0.1 mol/l of glycinebuffer, pH 3 with 2 mol/l of NaCl).

The eluate that contains bonded protein was diafiltered from 0.1% ofaqueous trifluoroacetic acid solution and first subjected to apreparative Reversed Phase HPLC separation analogously to the isolationof chemotactic peptides (cf. J. M. SCHRÖDER, (1997) Methods inEnzymology, Vol. 288, pp. 266-296). 20 μl of the respective fractionswas freeze-dried, taken up in 5 μl of a 0.1 percent aqueous acetic acidsolution and analyzed with the aid of a plate diffusion test system (seeExample 3) (for the identification of antimicrobial or antibioticproteins) analogously to M. E. SELSTED (1993) J. Biol. Chem., Vol. 268,pp. 6641-6648 with respect to the presence of antimicrobial peptides(with Staph. aureus or E. coli as test bacteria).

Antimicrobially active proteins that were eluted with 37% acetonitrilewere then subjected—analogously to the isolation of chemotactic peptides(J.-M. SCHRÖDER, (1977) Methods in Enzymology, Vol. 288, pp. 266-296)—toa micro-mono S-HPLC separation with the aid of the Smart-HPLC system.—SAP-3 was eluted with 0.79 mol/l of NaCl.

A subsequent micro-reversed phase-HPLC analysis with the aid of a C-18RP column yielded a protein peak that is eluted with 38% acetonitrile,which after SDS-gel electrophorese (performed according to the method ofJ. M. SCHRÖDER, (1997) Methods in Enzymology, Vol. 288, pp. 266-296)yielded an individual protein band corresponding to the mobility ofabout 6 kDa.

2.2 Sequencing of Fragments

Sequencing tests yielded only the amino-terminal sequence:

Gly Ile Ile Asn Thr Leu Gln Lys Tyr Tyr Cys Arg                  5                  10Val Arg Gly Gly Arg Cys Ala Val Leu Ser Cys Leu         15                  20 Pro Lys Glu Glu Gln Ile Gly Lys 25                  30      32(amino acid residues 1-32 of SEQ ID NO: 2)

2.3 Biological Activity of SAP-3 MIC

Antimicrobial activity from Staph aureus: <100 μg/ml from E. coli:  <20μg/ml

Example 3 3. Determination of Antimicrobial Activity 3.1 Cultivation ofMicroorganisms

The following microorganisms were used for the tests:

-   -   Escherichia coli (E. coli)—ATCC (American Type Culture        Collection) No. 11303    -   Pseudomonas aeruginosa—ATCC No. 15442    -   Staphylococcus aureus (clinical isolates from the dermatological        hospital of Kiel)    -   Staphylococcus epidermidis (clinical isolates from the        dermatological hospital of Kiel)    -   Candida albicans (clinical isolates from the dermatological        hospital of Kiel)

The microorganisms were cultivated on Trypticase-soy-broth (TSB) agarplates at 37° C. If they were not needed for a long time, they werestored at 4° C. For the tests, in each case a single colony of thecorresponding microorganisms was inoculated in 40 ml of TSB medium andincubated overnight at 37° C. while being shaken (250 rpm). To quantifythe microorganisms, the optical density of the overnight cultures wasmeasured at 620 nm (OD₆₂₀), and the colony number was determined byflattening out corresponding dilution stages.

3.2 Plate Diffusion Test

To study as quickly and sensitively as possible the antimicrobial actionof fractions of the individual chromatographic purification steps (cf.3.3), a radial plate diffusion test (cf. Hiemstra et al., 1993) wasused.

To obtain bacteria from a logarithmic growth phase, 20 μl of a 40 mlovernight culture of E. coli or Staphyloccus aureus in 8 ml oftrypticase-soy broth (TSB) was inoculated and incubated for 3.5 hours at37° C. The bacteria were then centrifuged off for 10 minutes at 1000 g,washed with 4° C. sodium-phosphate buffer (10 mmol, pH 7.4), resuspendedin 1 ml of sodium-phosphate buffer and quantified by determination ofOD₆₂₀. About 1×10⁶ bacteria were then added to 8 ml of preheated (42°C.) agarose medium, which consisted of 1% agarose in sodium-phosphatebuffer+1% TSB medium (v/v)+0.03% Tween 80 (v/v). After feeding thisagarose medium that is mixed with bacteria into a Petri dish (ø=10 cm;Sarstedt, Newton) and subsequent cooling at room temperature, holes witha 3 mm diameter were punched into the now solidified agarose layer. 5 μlof the substance in 0.01% acetic acid that was to be tested was thenadded to these holes.

After the incubation time was completed, the agarose layer was coveredwith a layer of 42° C. 2×TSB medium+1% agarose and incubated at 37° C.After about 20 to 24 hours, the inhibiting zones of the antimicrobial orantibiotic fractions could be clearly detected in the bacteria bed. Therelative antimicrobial or antibiotic activities were determined by therespective diameter of the inhibiting zones.

3.3 Liquid Culture Test

To be able to assess the dose-dependent range of action of anantimicrobial or antibiotic protein, a liquid culture testing system wasused (cf. Ganz et al., 1985).

About 10 μl of a 1×10⁷/ml dilution of the corresponding microorganismsin sodium-phosphate buffer (cf. 3.1) was added to 80 μl ofsodium-phosphate buffer together with 1.25% TSB medium (v/v). 10 μl of0.01% acetic acid was added with the corresponding concentrations of theantimicrobial or antibiotic protein (100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5μg/ml, and 6.25 μg/ml).

These batches were incubated in a 96-hole plate (Becton Dickinson,Heidelberg) for 3 hours at 37° C. while being shaken lightly (150 rpm).After the incubation time, tenfold dilution series were made from 50 μlof each of the batches with sodium-phosphate buffer and flattened out(100 μl) in each case in 3 parallel lines on TSB-agar plates. After 24to 36 hours of growth, the colonies were counted out.

As a control, one batch each was flattened out only with 10 μl of 0.01%acetic acid (without protein) once just before incubation and once after2 hours of incubation at 37° C.

4. Test Results

SAP-3 was incubated at the indicated concentrations at 37° C. for 3hours with 5·10⁴ KBE/ml (KBE=colony-forming units) of E. coli andStaphylococcus aureus in 100 μl of 10 mmol of sodium phosphate buffer(pH=7.4) together with 1% TSB (TSB=trypticase-soy broth). Theantimicrobial activity of SAP-3 was determined by counting out the KBEon the subsequent day. 100 μl of the respective batches in tenfolddilution stages was flattened out on TSB plates in advance. Then, theplates were incubated overnight at 37° C.

An LD₉₀ of 2.5-5 μg/ml is produced for E. coli and Staphylococcus aureus(LD₉₀=lethal dose of 90%; indicates the concentration range of therespective antimicrobial substances, in which the result is a 90%reduction of the colony-forming units that are used after three hours ofincubation with this antimicrobial substance).

SAP-2 was incubated at the indicated concentrations at 37° C. for 3hours with 1·10⁵ KBE/ml of the respective microorganisms in 100 μl of 10mmol of sodium phosphate buffer (pH=7.4) together with 1% TSB. Theantimicrobial activity of SAP-2 was determined by counting out the KBEon the subsequent day. 100 μl of the respective batches was flattenedout in tenfold dilution stages on TSB plates in advance. Then, theplates were incubated at 37° C. overnight.

There follows an LD₉₀ of 4-7.5 μg/ml for propionibacterium acnes; 7.5-15μg/ml for Staphylococcus aureus and Pseudomonas aeruginosa; in addition15-30 μg/ml for Candida albicans.

Example 5 Biochemical Characterization of Antimicrobial or AntibioticProteins with SDS-Polyacrylamide-Gel Electrophoresis (SDS-PAGE)

To determine the relative molecular weight, thetricine-SDS-polyacrylamide-gel electrophoresis was used (Schägger andJagow, 1987), which makes it possible to separate small proteins ofunder 10 kDA very efficiently.

The execution took place according to the protocol of the authors(Schägger and Jagow, 1987) in a vertical gel electrophoresis chamber,whereby a 16.5% polyacrylamide gel was used with a portion of 6%bisacrylamide and 6 M urea.

The samples were denatured before the application by 0.1 M DTT andboiling up. As a molecular size marker, the standard S-17 (Sigma, St.Louis, USA) was used. After to the course of the electrophoresis, thegel was subjected to silver coloration:

-   -   First, the gel was set for 30 minutes (30% ethanol, 10% glacial        acetic acid).    -   Then, a 30 minute incubation in “Farmer's reducer” solution was        carried out.    -   Then, the gel was washed with H₂O three times for 10 minutes and        colored in silver nitrate solution for 20 minutes.    -   Finally, a 10-15 minute incubation was carried out in developer        solution.    -   The development was stopped by 5% acetic acid, and the gel was        then photographed.

For a quick analysis of the HPLC fractions, the SDS-Page-Phast system(Pharmacia, Freiburg) was used with ready-to-use high-density gels(Pharmacia) according to information from the manufacturer. As sizemarkers, the S-17 markers of Sigma (see above) were used. The detectionof the separated molecules was carried out with the above-describedsilver coloration.

What is claimed is:
 1. An isolated cDNA, wherein the cDNA encodes aprotein comprising the amino acid sequence that is at least 95%identical to SEQ ID NO: 1 (SAP-2).
 2. The isolated cDNA according toclaim 1, whereby the cDNA codes for a mature protein.
 3. An isolatedcDNA, wherein the cDNA comprises the nucleotide sequence of SEQ ID NO: 5(cDNA-SAP-2) or a nucleotide sequence that is at least 95% identical toSEQ ID NO:
 5. 4. An isolated vector that contains a cDNA according toclaim 1 or 3, and a suitable promoter and optionally a suitableenhancer.
 5. An isolated eukaryotic or prokaryotic host cell transformedwith the vector according to claim
 4. 6. A bandage with at least oneprotein encoded by the isolated cDNA of claim 1 or
 3. 7. The isolatedcDNA of claim 1, wherein the isolated cDNA or DNA codes for an aminoacid sequence that is at least 98% identical to SEQ ID NO:
 1. 8. Theisolated cDNA of claim 3, wherein the isolated cDNA has the nucleotidesequence of SEQ ID NO: 5.